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LUND UNIVERSITY

Tissue repair in lung disorders

Andersson Sjöland, Annika

2009

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Citation for published version (APA):

Andersson Sjöland, A. (2009). Tissue repair in lung disorders. Lund University: Faculty of Medicine.

Total number of authors: 1

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From the Department of Clinical Sciences, Lund and

From the Department of Experimental Medical Science Lund University

Tissue repair in lung disorders

Annika Andersson Sjöland

AKADEMISK AVHANDLING

för avläggande av doktorsexamen i medicinsk vetenskap vid Medicinska Fakulteten, Lunds Universitet,

kommer att offentligen försvaras i Belfragesalen, BMC, Lund, fredagen den 11 december 2009, kl. 9.00.

Fakultetsopponent: Prof. Gunnar Pejler Uppsala Universitet

From the Department of Clinical Sciences, Lund and

From the Department of Experimental Medical Science Lund University

Tissue repair in lung disorders

Annika Andersson Sjöland

AKADEMISK AVHANDLING

för avläggande av doktorsexamen i medicinsk vetenskap vid Medicinska Fakulteten, Lunds Universitet,

kommer att offentligen försvaras i Belfragesalen, BMC, Lund, fredagen den 11 december 2009, kl. 9.00.

Fakultetsopponent: Prof. Gunnar Pejler Uppsala Universitet

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Tissue repair in lung disorders

Annika Andersson Sjöland

Tissue repair in lung disorders

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ISSN 1652-8220

ISBN 978-91-86443-11-5

Lund University, Faculty of Medicine Doctoral Dissertation Series 2009:122 Print by MEDIA-TRYCK Lund 2009

ISSN 1652-8220

ISBN 978-91-86443-11-5

Lund University, Faculty of Medicine Doctoral Dissertation Series 2009:122 Print by MEDIA-TRYCK Lund 2009

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Till Magnus,

Hannes & Kajsa

Till Magnus,

Hannes & Kajsa

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Contents

 List of Papers 9 Abbreviations 11 Introduction 13 Background 15 Lung Disorders 15

Structural Cells Involved in Tissue Repair 22 Extracellular Matrix Molecules in Tissue Repair 28

Aims 35

Methods 37

Subjects 37

General Histopathological Evaluation 39 Fibrocyte Characterization 39 Detection of Stromal Derived Factor 1/CXCL12 40 Vessel Identification 40 Fibroblast Culture 40 Fibroblast Characterization 40 Proteoglycan Assays 41 Migration Assays 41 Proliferation Assays 41 Results 43

Fibrocytes are a Potential Source of Lung Fibroblasts in Idiopathic Pulmonary Fibrosis (Paper I) 43 Fibrocytes are Associated with Vascular and Parenchymal Remodelling in Patients with Obliterative Bronchiolitis (Paper II) 46 Fibroblasts from Lung-transplanted Patients have Altered Proteoglycan and Proliferation Profiles Compared to Controls (Paper III) 47 Altered Matrix Production in the Distal Airways of Asthmatic and Atopic Individuals (Paper IV) 50

General Discussion 53

Future Perspectives 59

Summary in Swedish 61

Acknowledgements 63

Reference List 65

Appendix Paper I-IV

Contents

 List of Papers 9 Abbreviations 11 Introduction 13 Background 15 Lung Disorders 15

Structural Cells Involved in Tissue Repair 22 Extracellular Matrix Molecules in Tissue Repair 28

Aims 35

Methods 37

Subjects 37

General Histopathological Evaluation 39 Fibrocyte Characterization 39 Detection of Stromal Derived Factor 1/CXCL12 40 Vessel Identification 40 Fibroblast Culture 40 Fibroblast Characterization 40 Proteoglycan Assays 41 Migration Assays 41 Proliferation Assays 41 Results 43

Fibrocytes are a Potential Source of Lung Fibroblasts in Idiopathic Pulmonary Fibrosis (Paper I) 43 Fibrocytes are Associated with Vascular and Parenchymal Remodelling in Patients with Obliterative Bronchiolitis (Paper II) 46 Fibroblasts from Lung-transplanted Patients have Altered Proteoglycan and Proliferation Profiles Compared to Controls (Paper III) 47 Altered Matrix Production in the Distal Airways of Asthmatic and Atopic Individuals (Paper IV) 50

General Discussion 53

Future Perspectives 59

Summary in Swedish 61

Acknowledgements 63

Reference List 65

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List of Papers

Paper to defend:

I. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis.

Andersson-Sjöland A*, de Alba CG*, Nihlberg K, Becerril C, Ramírez R, Pardo A, Westergren-Thorsson G, Selman M.

Int J Biochem Cell Biol. 2008;40(10):2129-40. Epub 2008 Mar 11.

II. Fibrocytes are associated with vascular and parenchymal remodelling in patients with obliterative bronchiolitis

Annika Andersson-Sjöland, Jonas S Erjefält, Leif Bjermer, Leif Eriksson, Gunilla Westergren-Thorsson

Respiratory Research 2009, 10:103

III. Fibroblasts from lung-transplanted patients have altered proteoglycan and proliferation profiles compared to controls

A Follow-up Study of Lung Transplanted Patients – with Focus on Fibroblasts

Annika Andersson-Sjöland, Kristian Nihlberg, Lena Thiman , Leif Eriksson, Leif Bjermer, Gunilla Westergren-Thorsson,

Manuscript

IV. Altered matrix production in the distal airways of asthmatic and atopic individuals

Kristian Nihlberg, Annika Andersson-Sjöland, Ellen Tufvesson, Jonas S Erjefält Leif Bjermer, Gunilla Westergren-Thorsson,

Submitted to Thorax.

Paper I is reproduced with permission of Elsevier

Paper II is reproduced with permission of Biomed Central *These authors contributed equally to this work.

List of Papers

Paper to defend:

I. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis.

Andersson-Sjöland A*, de Alba CG*, Nihlberg K, Becerril C, Ramírez R, Pardo A, Westergren-Thorsson G, Selman M.

Int J Biochem Cell Biol. 2008;40(10):2129-40. Epub 2008 Mar 11.

II. Fibrocytes are associated with vascular and parenchymal remodelling in patients with obliterative bronchiolitis

Annika Andersson-Sjöland, Jonas S Erjefält, Leif Bjermer, Leif Eriksson, Gunilla Westergren-Thorsson

Respiratory Research 2009, 10:103

III. Fibroblasts from lung-transplanted patients have altered proteoglycan and proliferation profiles compared to controls

A Follow-up Study of Lung Transplanted Patients – with Focus on Fibroblasts

Annika Andersson-Sjöland, Kristian Nihlberg, Lena Thiman , Leif Eriksson, Leif Bjermer, Gunilla Westergren-Thorsson,

Manuscript

IV. Altered matrix production in the distal airways of asthmatic and atopic individuals

Kristian Nihlberg, Annika Andersson-Sjöland, Ellen Tufvesson, Jonas S Erjefält Leif Bjermer, Gunilla Westergren-Thorsson,

Submitted to Thorax.

Paper I is reproduced with permission of Elsevier

Paper II is reproduced with permission of Biomed Central *These authors contributed equally to this work.

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Additional papers

Pathological airway remodeling in airway in inflammation

Westergren-Thorsson G, Larsen K, Nihlberg K, Andersson-Sjöland A, Hallgren O, Marko-Varga G, Bjermer L,

Submitted to Clinical Respiratory Journal, 2009

Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild asthma.

Larsen K, Tufvesson E, Malmström J, Mörgelin M, Wildt M, Andersson A, Lindström A, Malmström A, Löfdahl CG, Marko-Varga G, Bjermer L, Westergren-Thorsson G.

Am J Respir Crit Care Med. 2004 Nov 15;170(10):1049-56. Epub 2004 Jul 15.

The role of glycosaminoglycan binding of staphylococci in attachment to eukaryotic host cells.

Fallgren C, Andersson A, Ljungh A.

Curr Microbiol. 2001 Jul;43(1):57-63.

Additional papers

Pathological airway remodeling in airway in inflammation

Westergren-Thorsson G, Larsen K, Nihlberg K, Andersson-Sjöland A, Hallgren O, Marko-Varga G, Bjermer L,

Submitted to Clinical Respiratory Journal, 2009

Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild asthma.

Larsen K, Tufvesson E, Malmström J, Mörgelin M, Wildt M, Andersson A, Lindström A, Malmström A, Löfdahl CG, Marko-Varga G, Bjermer L, Westergren-Thorsson G.

Am J Respir Crit Care Med. 2004 Nov 15;170(10):1049-56. Epub 2004 Jul 15.

The role of glycosaminoglycan binding of staphylococci in attachment to eukaryotic host cells.

Fallgren C, Andersson A, Ljungh A.

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Abbreviations

BAL bronchoalveolar lavage BOS bronchiolitis obliterans syndrome CF cystic fibrosis

CMV cytomegalovirus CS chondriotin sulphate

COPD chronic obstructive pulmonary disease DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco’s Eagle’s minimal essential medium DS dermatan sulphate

ECM extracellular matrix EGF epidermal growth factor

EMT epithelial-mesenchymal transition FGF fibroblast growth factor

FEV1 forced expiratory volume in 1 second

HIF hypoxia-induced factor HS heparin sulphate GAG glycosaminoglycan IFN- interferon-

IL interleukin IPF idiopathic pulmonary fibrosis MAPK mitogen-activated protein kinases MHC major histocompatibility complex MMP matrix metalloproteinase OB obliterative bronchiolitis PDGF platelet-derived growth factor SAPK/JNK stress-activated protein kinase /

jun N-terminal kinases SDF-1 / CXCL12 stromal cell-derived factor-1 /

chemokine ligand 12 -SMA -smooth muscle cell

TGF- transforming growth factor- TNF- tumor necrosis factor-

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor vWF von Willebrand factor

ZO zona occludens

Abbreviations

BAL bronchoalveolar lavage BOS bronchiolitis obliterans syndrome CF cystic fibrosis

CMV cytomegalovirus CS chondriotin sulphate

COPD chronic obstructive pulmonary disease DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco’s Eagle’s minimal essential medium DS dermatan sulphate

ECM extracellular matrix EGF epidermal growth factor

EMT epithelial-mesenchymal transition FGF fibroblast growth factor

FEV1 forced expiratory volume in 1 second

HIF hypoxia-induced factor HS heparin sulphate GAG glycosaminoglycan IFN- interferon-

IL interleukin IPF idiopathic pulmonary fibrosis MAPK mitogen-activated protein kinases MHC major histocompatibility complex MMP matrix metalloproteinase OB obliterative bronchiolitis PDGF platelet-derived growth factor SAPK/JNK stress-activated protein kinase /

jun N-terminal kinases SDF-1 / CXCL12 stromal cell-derived factor-1 /

chemokine ligand 12 -SMA -smooth muscle cell

TGF- transforming growth factor- TNF- tumor necrosis factor-

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor vWF von Willebrand factor

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Introduction

Tissue repair processes and remodelling are ongoing processes in all types of wound healing. In healthy subjects, the primary role of the extracellular matrix (ECM) is to provide tissues with specific mechanical properties, and to serve as a structural framework for cell attachment and migration. An ongoing tissue repair can result in fibrosis, regarded as an abnormal wound-healing process. In the lung the fibrosis can be localised in the central part of the lung, or in the distal alveolar parenchymal part, or something in-between, in the small airways. In this thesis, we have included three different patient groups believed to differ somewhat in primary site of fibrotic deposition. In asthma, the basement membrane, which is located below the epithelial layer, is thickened because of accumulation of collagens and proteoglycans. Idiopathic pulmonary fibrosis (IPF) is characterised by fibroblastic foci that are in demarcated areas rich in ECM and proteoglycans but with few cells. In obliterative bronchiolitis (OB), the small airways are obliterated with ECM where the proteoglycans function as staples to attach the connective tissue. In OB, the parenchymal part of the lung is also involved with a thickening of the alveolar septa. The structural changes will be discussed in more detail later in this thesis.

The above-mentioned disorders are chronic diseases with remodelling of both the airways and the pulmonary vessels. The remodelling processes have many differences but, surprisingly, also many similarities even though the underlying pathophysiologies are different. Remodelling usually starts with an epithelial injury that later gives rise to structural changes in the airways and in the lung architecture, featuring lung function decrease and chronic airway symptoms. Tissue repair and inflammation often interact in a dynamic and parallel manner. The players at both the cellular and the molecular levels are dependent on both the type of disease and the disease state of the patient. Inflammation is a process that can be preceded by infections. Depending of what kind of stimuli triggered the inflammation, the response of the immune system is regulated in different ways. The immune system can be divided into an innate part on the one hand, including barriers to the surroundings, eosinophils, neutrophils, macrophages, and natural killer cells, and an adaptive part on the other, including T-lymphocytes and B-lymphocytes.

One of the diseases that has both inflammatory and remodelling features is asthma. Asthma is a widespread disease; today, 8% of the adult population in Sweden suffers from asthma (1). Another condition in which both inflammation and

Introduction

Tissue repair processes and remodelling are ongoing processes in all types of wound healing. In healthy subjects, the primary role of the extracellular matrix (ECM) is to provide tissues with specific mechanical properties, and to serve as a structural framework for cell attachment and migration. An ongoing tissue repair can result in fibrosis, regarded as an abnormal wound-healing process. In the lung the fibrosis can be localised in the central part of the lung, or in the distal alveolar parenchymal part, or something in-between, in the small airways. In this thesis, we have included three different patient groups believed to differ somewhat in primary site of fibrotic deposition. In asthma, the basement membrane, which is located below the epithelial layer, is thickened because of accumulation of collagens and proteoglycans. Idiopathic pulmonary fibrosis (IPF) is characterised by fibroblastic foci that are in demarcated areas rich in ECM and proteoglycans but with few cells. In obliterative bronchiolitis (OB), the small airways are obliterated with ECM where the proteoglycans function as staples to attach the connective tissue. In OB, the parenchymal part of the lung is also involved with a thickening of the alveolar septa. The structural changes will be discussed in more detail later in this thesis.

The above-mentioned disorders are chronic diseases with remodelling of both the airways and the pulmonary vessels. The remodelling processes have many differences but, surprisingly, also many similarities even though the underlying pathophysiologies are different. Remodelling usually starts with an epithelial injury that later gives rise to structural changes in the airways and in the lung architecture, featuring lung function decrease and chronic airway symptoms. Tissue repair and inflammation often interact in a dynamic and parallel manner. The players at both the cellular and the molecular levels are dependent on both the type of disease and the disease state of the patient. Inflammation is a process that can be preceded by infections. Depending of what kind of stimuli triggered the inflammation, the response of the immune system is regulated in different ways. The immune system can be divided into an innate part on the one hand, including barriers to the surroundings, eosinophils, neutrophils, macrophages, and natural killer cells, and an adaptive part on the other, including T-lymphocytes and B-lymphocytes.

One of the diseases that has both inflammatory and remodelling features is asthma. Asthma is a widespread disease; today, 8% of the adult population in Sweden suffers from asthma (1). Another condition in which both inflammation and

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remodelling are important characteristics is chronic rejection after organ transplantation. The first lung transplantation was done in 1983 in Toronto, Canada (2) and today around 40 lung transplantations are performed annually in Sweden (about 15 at the University hospital in Lund and about 25 at Sahlgrenska University Hospital in Göteborg). Unfortunately, up to 60% of the transplanted patients develop bronchiolitis obliterans syndrome (BOS), as a sign of chronic rejection. IPF is a disease where the cause still is unclear and the possibilities of treatments are limited. Some patients with IPF will eventually be candidates for lung transplantation. However, the most common disorders today, leading to transplantations are chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (3).

The aims of the work for this thesis were to determine whether fibrocytes, which are progenitors of fibroblasts, are of importance in tissue remodeling. A second aim was to further characterise factors involved in the recruitment of fibrocytes from the bloodstream to specific areas of the lung. A third aim was to identify phenotypes of fibroblasts that are associated with tissue repair.

remodelling are important characteristics is chronic rejection after organ transplantation. The first lung transplantation was done in 1983 in Toronto, Canada (2) and today around 40 lung transplantations are performed annually in Sweden (about 15 at the University hospital in Lund and about 25 at Sahlgrenska University Hospital in Göteborg). Unfortunately, up to 60% of the transplanted patients develop bronchiolitis obliterans syndrome (BOS), as a sign of chronic rejection. IPF is a disease where the cause still is unclear and the possibilities of treatments are limited. Some patients with IPF will eventually be candidates for lung transplantation. However, the most common disorders today, leading to transplantations are chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (3).

The aims of the work for this thesis were to determine whether fibrocytes, which are progenitors of fibroblasts, are of importance in tissue remodeling. A second aim was to further characterise factors involved in the recruitment of fibrocytes from the bloodstream to specific areas of the lung. A third aim was to identify phenotypes of fibroblasts that are associated with tissue repair.

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Background

Lung Disorders

This thesis concentrates on tissue repair, and remodelling in lung disorders such as IPF, OB after lung or bone marrow transplantation, and asthma is discussed. The origins of these disorders are different, but they have the common denominator that ECM deposition changes the lung structure and causes deterioration of the tissue, and thereby of lung function.

Idiopathic Pulmonary Fibrosis

IPF is a chronic disease that is usually lethal, is progressive, and is of unknown cause (4). The disease is by definition grouped as an idiopathic interstitial pneumonia, but is distinguished histologically from other diseases in the group because of its characteristic fibroblast foci of proliferative and matrix-producing fibroblasts and myofibroblasts. The diagnosis of IPF can be established after surgical lung biopsies with preoperative high-resolution computed tomography to show specific abnormal regions of the lung (5). IPF is more common in men than in women, and the age at onset of the disease is around 60 years. The treatments for IPF are focused on inflammation, fibrosis, and the immune response but unfortunately the effects of pharmacological treatment are limited. Lung transplantation has been shown to be a good treatment for some IPF patients, even though this solution is only possible if the patients, excluding lung status are healthy enough to undergo transplantation (6).

Even though the cause of the fibrosis is unknown, there are some substances that have been shown to be associated with the disease, such as asbestos, substances associated with agriculture and livestock, metal dust, and cigarette smoke (7). Cigarette smoke, which reduces the length of telomeres, could be an important pathogenetic factor in IPF (8). In families with IPF, 8% of individuals have a mutation in genes that result in short telomeres (9).

Many cell types are of importance for the pathology behind IPF, but fibroblasts with their ability to produce matrix molecules are of special interest. In tissues from patients with IPF, fibroblastic foci have been identified as discrete areas rich in ECM but with few cells (Figure 1). The cells of the fibroblast foci are arranged in an outstretched and parallel arrangement relative to the other cells and to the alveolar septa (10). Fibroblasts in the lungs of IPF patients could come from epithelial cells, from fibrocytes recruited from the bone marrow, or from proliferation of residual fibroblasts. For the origin of fibroblasts derived from

Background

Lung Disorders

This thesis concentrates on tissue repair, and remodelling in lung disorders such as IPF, OB after lung or bone marrow transplantation, and asthma is discussed. The origins of these disorders are different, but they have the common denominator that ECM deposition changes the lung structure and causes deterioration of the tissue, and thereby of lung function.

Idiopathic Pulmonary Fibrosis

IPF is a chronic disease that is usually lethal, is progressive, and is of unknown cause (4). The disease is by definition grouped as an idiopathic interstitial pneumonia, but is distinguished histologically from other diseases in the group because of its characteristic fibroblast foci of proliferative and matrix-producing fibroblasts and myofibroblasts. The diagnosis of IPF can be established after surgical lung biopsies with preoperative high-resolution computed tomography to show specific abnormal regions of the lung (5). IPF is more common in men than in women, and the age at onset of the disease is around 60 years. The treatments for IPF are focused on inflammation, fibrosis, and the immune response but unfortunately the effects of pharmacological treatment are limited. Lung transplantation has been shown to be a good treatment for some IPF patients, even though this solution is only possible if the patients, excluding lung status are healthy enough to undergo transplantation (6).

Even though the cause of the fibrosis is unknown, there are some substances that have been shown to be associated with the disease, such as asbestos, substances associated with agriculture and livestock, metal dust, and cigarette smoke (7). Cigarette smoke, which reduces the length of telomeres, could be an important pathogenetic factor in IPF (8). In families with IPF, 8% of individuals have a mutation in genes that result in short telomeres (9).

Many cell types are of importance for the pathology behind IPF, but fibroblasts with their ability to produce matrix molecules are of special interest. In tissues from patients with IPF, fibroblastic foci have been identified as discrete areas rich in ECM but with few cells (Figure 1). The cells of the fibroblast foci are arranged in an outstretched and parallel arrangement relative to the other cells and to the alveolar septa (10). Fibroblasts in the lungs of IPF patients could come from epithelial cells, from fibrocytes recruited from the bone marrow, or from proliferation of residual fibroblasts. For the origin of fibroblasts derived from

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epithelial cells the level of TGF-, a stimulator of epithelial-mesenchymal transition (EMT), is known to be increased in IPF lungs (11). Furthermore, EMT is regulated by Gremlin, which is an inhibitor of bone morphogenetic proteins, and is up-regulated in IPF so that epithelial cells are more sensitive to TGF- (12). Another possible origin of the fibroblasts in IPF is recruitment of fibrocytes from the bone marrow. Fibrocytes as a possible source of fibroblasts are discussed in detail below, but it needs to be mentioned that the chemokine stromal cell-derived factor-1/chemokine ligand 12 (SDF-1/CXCL12) is up-regulated in both plasma and bronchoalveolar lavage (BAL) fluid in patients with IPF (Paper 1). Concerning vessel remodelling in IPF, it is of interest that the concentration of VEGF, a growth factor involved in vessel formation, is reduced in BAL fluid and it is not expressed in fibroblastic foci. In an in vitro study, it has also been shown that endothelial tubule formation is suppressed in the presence of IPF lung homogenates fromIPF lung (13).

Figure 1. Fibroblastic foci are a characteristic feature of tissue from patients with idiopathic pulmonary fibrosis. The foci are identified as demarcated areas rich in ECM but with few cells. The cells in the fibroblast foci are outstretched and aligned parallel to the other cells and to the alveolar septa. A fibroblast focus can be seen here after Gomori’s trichrome staining (see dashed line). Original magnification: 20×.

epithelial cells the level of TGF-, a stimulator of epithelial-mesenchymal transition (EMT), is known to be increased in IPF lungs (11). Furthermore, EMT is regulated by Gremlin, which is an inhibitor of bone morphogenetic proteins, and is up-regulated in IPF so that epithelial cells are more sensitive to TGF- (12). Another possible origin of the fibroblasts in IPF is recruitment of fibrocytes from the bone marrow. Fibrocytes as a possible source of fibroblasts are discussed in detail below, but it needs to be mentioned that the chemokine stromal cell-derived factor-1/chemokine ligand 12 (SDF-1/CXCL12) is up-regulated in both plasma and bronchoalveolar lavage (BAL) fluid in patients with IPF (Paper 1). Concerning vessel remodelling in IPF, it is of interest that the concentration of VEGF, a growth factor involved in vessel formation, is reduced in BAL fluid and it is not expressed in fibroblastic foci. In an in vitro study, it has also been shown that endothelial tubule formation is suppressed in the presence of IPF lung homogenates fromIPF lung (13).

Figure 1. Fibroblastic foci are a characteristic feature of tissue from patients with idiopathic pulmonary fibrosis. The foci are identified as demarcated areas rich in ECM but with few cells. The cells in the fibroblast foci are outstretched and aligned parallel to the other cells and to the alveolar septa. A fibroblast focus can be seen here after Gomori’s trichrome staining (see dashed line). Original magnification: 20×.

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Lung Transplantation and Fibro-obliteration

Lung transplantation is a treatment available for end-stage lung disease in patients with COPD, CF, lymphangioleiomyomatosis, and fibrosing interstitial lung disorders (ILD) such as IPF. Depending on the diagnosis, single or bilateral lung transplantation is performed. The number of transplantations is still limited by the number of donors available. At the time of transplantation, immunosuppressant treatment is started and continues for the rest of the patient’s life.

During the operation, the bronchial circulation is not restored for technical reasons. A Danish study has, however, shown that restoration of bronchial artery revascularization delays the onset of BOS (14). Also, the vagal nerve is often injured during the transplantation and results in deterioration in the patterns of breathing and coughing, which can give rise to an increased risk of pneumonia and aspiration of gastroesophageal secretions, and of developing BOS. However, the cough reflex is restored 12 months after the transplantation (15).

Without pharmacological immunosuppressant treatment, transplanted lungs should be rejected in a few days in an alloimmune rejection. The treatment today often combines immune-suppressive drugs such as cyclosporine A and azathioprine with steorids.

Chronic rejection is a common consequence of lung transplantation (affecting 60% of those transplanted) (3) (Figure 2). The tissue process starts with lymphocyte infiltration in the submucosa and injury of the mucosa and epithelial cell layer, which results in recruitment of ECM-producing fibroblasts or the progenitor cells, fibrocytes. Histologically, the rejection is seen as an ECM plug with few fibroblasts in the bronchioles (16-18). Clinically, the rejection is apparent as reduced lung function, and cough and dyspnea are common symptoms. Table 1 covers the criteria for the diagnosis of BOS (19). For recommendations concerning choice of spirometric equipment, confounding conditions, definition of baseline, and BOS stages, see reference (16).

Lung Transplantation and Fibro-obliteration

Lung transplantation is a treatment available for end-stage lung disease in patients with COPD, CF, lymphangioleiomyomatosis, and fibrosing interstitial lung disorders (ILD) such as IPF. Depending on the diagnosis, single or bilateral lung transplantation is performed. The number of transplantations is still limited by the number of donors available. At the time of transplantation, immunosuppressant treatment is started and continues for the rest of the patient’s life.

During the operation, the bronchial circulation is not restored for technical reasons. A Danish study has, however, shown that restoration of bronchial artery revascularization delays the onset of BOS (14). Also, the vagal nerve is often injured during the transplantation and results in deterioration in the patterns of breathing and coughing, which can give rise to an increased risk of pneumonia and aspiration of gastroesophageal secretions, and of developing BOS. However, the cough reflex is restored 12 months after the transplantation (15).

Without pharmacological immunosuppressant treatment, transplanted lungs should be rejected in a few days in an alloimmune rejection. The treatment today often combines immune-suppressive drugs such as cyclosporine A and azathioprine with steorids.

Chronic rejection is a common consequence of lung transplantation (affecting 60% of those transplanted) (3) (Figure 2). The tissue process starts with lymphocyte infiltration in the submucosa and injury of the mucosa and epithelial cell layer, which results in recruitment of ECM-producing fibroblasts or the progenitor cells, fibrocytes. Histologically, the rejection is seen as an ECM plug with few fibroblasts in the bronchioles (16-18). Clinically, the rejection is apparent as reduced lung function, and cough and dyspnea are common symptoms. Table 1 covers the criteria for the diagnosis of BOS (19). For recommendations concerning choice of spirometric equipment, confounding conditions, definition of baseline, and BOS stages, see reference (16).

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Figure 2. Histologically, obliterative bronchiolitis can be seen as fibro-proliferative intraluminal plugs with few fibroblasts in the terminal respiratory bronchioles. The dashed line indicates the plug, while the two arrows show neo-lumina. Original magnification: 20×.

BOS 0 FEV1 > 90% of baseline and

FEF25–75 > 75% of baseline

BOS 0p FEV1 81–90% of baseline and/or

FEF25–75  75% of baseline

BOS 1 FEV1 66–80% of baseline

BOS 2 FEV1 51–65% of baseline

BOS 3 FEV1  50% of baseline

BOS = bronchiolitis obliterans syndrome, FEF = forced expiratory flow, FEV1 = forced expiratory volume in 1 second

Table 1. Classification system for bronchiolitis obliterans. Adapted from Estenne and Hertz 2002.

Figure 2. Histologically, obliterative bronchiolitis can be seen as fibro-proliferative intraluminal plugs with few fibroblasts in the terminal respiratory bronchioles. The dashed line indicates the plug, while the two arrows show neo-lumina. Original magnification: 20×.

BOS 0 FEV1 > 90% of baseline and

FEF25–75 > 75% of baseline

BOS 0p FEV1 81–90% of baseline and/or

FEF25–75  75% of baseline

BOS 1 FEV1 66–80% of baseline

BOS 2 FEV1 51–65% of baseline

BOS 3 FEV1  50% of baseline

BOS = bronchiolitis obliterans syndrome, FEF = forced expiratory flow, FEV1 = forced expiratory volume in 1 second

Table 1. Classification system for bronchiolitis obliterans. Adapted from Estenne and Hertz 2002.

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There have been a number of articles addressing risk factors for BOS, most commonly mentioned are acute cellular rejection, human leukocyte antigen (HLA) mismatch, infections by cytomegalovirus (CMV) or lymphocytic bronchitis, and female donor to male recipient (16;20). The pathogenesis of BOS involves both non-alloimmune mechanisms such as infections, ischaemia, and gastro-oesophageal reflux and alloimmune mechanisms such as acute rejection and lymphocytic bronchiolitis (19). Which of the immune responses (type 1, the cell-mediated response; type 2, driven by cytotoxic T-lymphocytes (3); or type 17, the autoimmune response (21)) causes the rejection is still under debate. The growth factors involved in the fibro-proliferative phase of the chronic rejection in particular are platelet-derived growth factor (PDGF) (22) and TGF- (23), which are known to up-regulate ECM deposition. Also, the size of vessels is increased in patients with BOS (20) (Paper 2) and this could be the result of local hypoxia, which promotes angiogenesis.

To prevent BOS attempts with a special double transplantation has shown promising results. At the same time as the lung transplantation occurs, it is combined with infusion of bone marrow from the same donor as the lung came from. The theory behind the double transplantation is that the recipient’s immune cells are modulated, and the results show a lower incidence of OB compared to lung transplantation alone. Interestingly, the numbers of acute cellular rejections were found to be the same in a group of doubly transplanted subjects as in a group of subjects who only underwent lung transplantation. The outcomes of the studies in the field are difficult to interpret because of the small size of patient groups and the lack of matched control groups (24).

The treatment of BOS is unfortunately limited to changes in the immunosuppressive medication, both concerning dose and type of therapy (3). Re-transplantation is an alternative treatment for BOS, even though re-Re-transplantation is associated with a higher mortality than initial transplantation (25). A five-year follow-up study of patients with BOS after initial transplantation showed that over 60% were alive even though this patient group had an elevated risk of developing BOS once again (25;26).

Bone Marrow / Haematopoietic Stem Cell Transplantation

A number of late complications such as pulmonary, cardiac, and renal complications are known to occur after bone marrow transplantation (27). The pulmonary complications occur at a frequency of 2–11% of the adult cases and 8% of the paediatric cases (28-31). As with OB after lung transplantation, it is mainly the small airways that are obliterated; and the risk factors for development of fibrosis are infections, smoking, cytotoxic therapy, irradiation, and chronic graft-versus-host disease (32;33). The histological characteristics of OB after bone marrow/haematopoietic stem cell transplantation are similar to those after lung

There have been a number of articles addressing risk factors for BOS, most commonly mentioned are acute cellular rejection, human leukocyte antigen (HLA) mismatch, infections by cytomegalovirus (CMV) or lymphocytic bronchitis, and female donor to male recipient (16;20). The pathogenesis of BOS involves both non-alloimmune mechanisms such as infections, ischaemia, and gastro-oesophageal reflux and alloimmune mechanisms such as acute rejection and lymphocytic bronchiolitis (19). Which of the immune responses (type 1, the cell-mediated response; type 2, driven by cytotoxic T-lymphocytes (3); or type 17, the autoimmune response (21)) causes the rejection is still under debate. The growth factors involved in the fibro-proliferative phase of the chronic rejection in particular are platelet-derived growth factor (PDGF) (22) and TGF- (23), which are known to up-regulate ECM deposition. Also, the size of vessels is increased in patients with BOS (20) (Paper 2) and this could be the result of local hypoxia, which promotes angiogenesis.

To prevent BOS attempts with a special double transplantation has shown promising results. At the same time as the lung transplantation occurs, it is combined with infusion of bone marrow from the same donor as the lung came from. The theory behind the double transplantation is that the recipient’s immune cells are modulated, and the results show a lower incidence of OB compared to lung transplantation alone. Interestingly, the numbers of acute cellular rejections were found to be the same in a group of doubly transplanted subjects as in a group of subjects who only underwent lung transplantation. The outcomes of the studies in the field are difficult to interpret because of the small size of patient groups and the lack of matched control groups (24).

The treatment of BOS is unfortunately limited to changes in the immunosuppressive medication, both concerning dose and type of therapy (3). Re-transplantation is an alternative treatment for BOS, even though re-Re-transplantation is associated with a higher mortality than initial transplantation (25). A five-year follow-up study of patients with BOS after initial transplantation showed that over 60% were alive even though this patient group had an elevated risk of developing BOS once again (25;26).

Bone Marrow / Haematopoietic Stem Cell Transplantation

A number of late complications such as pulmonary, cardiac, and renal complications are known to occur after bone marrow transplantation (27). The pulmonary complications occur at a frequency of 2–11% of the adult cases and 8% of the paediatric cases (28-31). As with OB after lung transplantation, it is mainly the small airways that are obliterated; and the risk factors for development of fibrosis are infections, smoking, cytotoxic therapy, irradiation, and chronic graft-versus-host disease (32;33). The histological characteristics of OB after bone marrow/haematopoietic stem cell transplantation are similar to those after lung

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transplantation (Figure 2). The symptoms are cough, dyspnea, and wheezing. The clinical OB is defined as forced expiratory volume in 1 second (FEV1)/ forced

vital capacity ratio < 0.7, FEV1 < 75% of predictedvalue, evidence of air trapping

or small airway thickening, and absence of infection (34). The treatments are limited to immunosuppressive drugs and corticosteroids or lung transplantation (31;35-37). For OB after lung transplantation, the outcome is poor (33).

Asthma

Also in asthma remodelling and accumulation of ECM are histological features. While asthma historically was regarded as a single disease it has during the last years been clear that asthma is a very heterogeneous disorder, rather a syndrome, including different clinical phenotypes (38). The diagnosis of asthma is often based on symptoms such as wheezing, coughing, shortness of breath, and chest tightness with objective measures as variable airflow obstruction and bronchial hyperresponsiveness (39). The different phenotypes of asthma are based on: (1) clinical observations such as frequency of exacerbations, air flow restriction, resistance to pharmaceuticals (corticosteroids), age at onset (40-42); (2) determining the trigger-factor such as allergens or exercise (43;44); and (3) determining the number and localisation of inflammatory cells such as eosinophils and neutrophils (45;46). There may also be some overlap between the different phenotypes of asthma.

The complexity of the disease and phenotypes has made it difficult to implicate genes in asthma. However, one gene (ADAM33) with polymorphism in asthma patients has been identified; it codes for a Zn+-dependent matrix metalloproteinase (MMP). ADAM33 is expressed in mesenchymal cells such as fibroblasts and smooth muscle cells (47;48).

Many cell types with different features are involved in asthma. The structural cells that are involved in asthma are for example epithelial cells, smooth muscle cells, and (myo)fibroblasts. The epithelial layer forms a structural barrier between the air space and the tissue of the lung. Some of the known risk factors in asthma, such as indoor and outdoor allergens, tobacco smoke, and air pollution, have their first contact with the patient by way of the epithelium (49). In studies of biopsies from asthmatic patients, a correlation has been found between the degree of epithelial loss and the degree of airway reactivity (50). The main function of the muscle cells are to contract tissue. Both the smooth muscle area and the size of the smooth muscle cells increase with the severity of asthma (51). Smooth muscle cells are capable of proliferating, migrating, producing pro-inflammatory cytokines, and forming new muscle with change of phenotype and function (52-54). Even though other cell types are capable of producing ECM molecules, the fibroblast is the main producer. Fibroblasts, myofibroblasts, the ECM, and collagen are described below in more detail.

transplantation (Figure 2). The symptoms are cough, dyspnea, and wheezing. The clinical OB is defined as forced expiratory volume in 1 second (FEV1)/ forced

vital capacity ratio < 0.7, FEV1 < 75% of predictedvalue, evidence of air trapping

or small airway thickening, and absence of infection (34). The treatments are limited to immunosuppressive drugs and corticosteroids or lung transplantation (31;35-37). For OB after lung transplantation, the outcome is poor (33).

Asthma

Also in asthma remodelling and accumulation of ECM are histological features. While asthma historically was regarded as a single disease it has during the last years been clear that asthma is a very heterogeneous disorder, rather a syndrome, including different clinical phenotypes (38). The diagnosis of asthma is often based on symptoms such as wheezing, coughing, shortness of breath, and chest tightness with objective measures as variable airflow obstruction and bronchial hyperresponsiveness (39). The different phenotypes of asthma are based on: (1) clinical observations such as frequency of exacerbations, air flow restriction, resistance to pharmaceuticals (corticosteroids), age at onset (40-42); (2) determining the trigger-factor such as allergens or exercise (43;44); and (3) determining the number and localisation of inflammatory cells such as eosinophils and neutrophils (45;46). There may also be some overlap between the different phenotypes of asthma.

The complexity of the disease and phenotypes has made it difficult to implicate genes in asthma. However, one gene (ADAM33) with polymorphism in asthma patients has been identified; it codes for a Zn+-dependent matrix metalloproteinase (MMP). ADAM33 is expressed in mesenchymal cells such as fibroblasts and smooth muscle cells (47;48).

Many cell types with different features are involved in asthma. The structural cells that are involved in asthma are for example epithelial cells, smooth muscle cells, and (myo)fibroblasts. The epithelial layer forms a structural barrier between the air space and the tissue of the lung. Some of the known risk factors in asthma, such as indoor and outdoor allergens, tobacco smoke, and air pollution, have their first contact with the patient by way of the epithelium (49). In studies of biopsies from asthmatic patients, a correlation has been found between the degree of epithelial loss and the degree of airway reactivity (50). The main function of the muscle cells are to contract tissue. Both the smooth muscle area and the size of the smooth muscle cells increase with the severity of asthma (51). Smooth muscle cells are capable of proliferating, migrating, producing pro-inflammatory cytokines, and forming new muscle with change of phenotype and function (52-54). Even though other cell types are capable of producing ECM molecules, the fibroblast is the main producer. Fibroblasts, myofibroblasts, the ECM, and collagen are described below in more detail.

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In asthma patients, the inflammatory response leads to release of cytokines and interleukins which results in narrowing of the airway lumen, secretion of mucus, accumulation of ECM molecules, increased number and size of smooth muscle cells, and thickening of the bronchial wall (55) (Figure 3). Mast cells and basophils have granulas containing chymase and tryptase, eosinophils can release a number of cytokines, leukotrienes, prostaglandins and the number of all these cell type is associated with a more severe asthma phenotype. Other immune cells that are involved in asthma are neutrophils that attract inflammatory cells, T-cells and macrophages and dendritic cells which act as antigen presenting cells (56-59).

Figure 3. During an asthma attack, smooth muscles around the airways contract which gives a tightening of the lumen. This, together with swelling and inflammation with infiltration of immune cells and excessive secretion of mucus into the airways, gives an obstructed airway. Adapted from: Encyclopædia Britannica.

Asthma therapy has been based on bronchodilator agents, often combined with anti-inflammatory treatment. The bronchodilators, 2-agonists, act by binding to 2-adenoreceptors located on smooth muscle cells, epithelial cells, and immune cells and cause smooth muscle cells in particular to relax (60;61). The anti-inflammatory drugs, glucocorticoids, act by binding to the glucocorticoid receptor

In asthma patients, the inflammatory response leads to release of cytokines and interleukins which results in narrowing of the airway lumen, secretion of mucus, accumulation of ECM molecules, increased number and size of smooth muscle cells, and thickening of the bronchial wall (55) (Figure 3). Mast cells and basophils have granulas containing chymase and tryptase, eosinophils can release a number of cytokines, leukotrienes, prostaglandins and the number of all these cell type is associated with a more severe asthma phenotype. Other immune cells that are involved in asthma are neutrophils that attract inflammatory cells, T-cells and macrophages and dendritic cells which act as antigen presenting cells (56-59).

Figure 3. During an asthma attack, smooth muscles around the airways contract which gives a tightening of the lumen. This, together with swelling and inflammation with infiltration of immune cells and excessive secretion of mucus into the airways, gives an obstructed airway. Adapted from: Encyclopædia Britannica.

Asthma therapy has been based on bronchodilator agents, often combined with anti-inflammatory treatment. The bronchodilators, 2-agonists, act by binding to 2-adenoreceptors located on smooth muscle cells, epithelial cells, and immune cells and cause smooth muscle cells in particular to relax (60;61). The anti-inflammatory drugs, glucocorticoids, act by binding to the glucocorticoid receptor

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in the cytoplasm and regulate gene transcription by activating anti-inflammatory genes (62).

Structural Cells Involved in Tissue Repair

Nearly all types of cells are involved in tissue repair. As mentioned at the beginning of this thesis, inflammation and remodelling work together in the disorders described here. In the following paragraphs, the main structural cell types that are involved in the remodelling process in tissue repair are presented.

Fibroblasts

Cytologically, fibroblasts are defined as elongated cells (Paper 4) with a typical marked rough endoplasmic reticulum and a typical Golgi apparatus that are common in cells that produce ECM and collagen (63) (Figure 4). The number of ECM molecules produced by fibroblasts are almost countless. The connective tissue from the fibroblasts surrounds the cells and provides a lattice to which the cells can bind and move along. Fibroblasts are in many cases a heterogeneous cell population and their behaviour is strongly dependent on and modulated by the surrounding environment, growth factors, and chemokines bound to the ECM.

10m

Figure 4. One of the structural cell types involved in tissue repair is the fibroblast. Fibroblasts are defined as elongated, cells with a typically marked rough endoplasmic reticulum. They can produce a number of extracellular matrix molecules. This cultured fibroblast was visualized by electron microscope.

in the cytoplasm and regulate gene transcription by activating anti-inflammatory genes (62).

Structural Cells Involved in Tissue Repair

Nearly all types of cells are involved in tissue repair. As mentioned at the beginning of this thesis, inflammation and remodelling work together in the disorders described here. In the following paragraphs, the main structural cell types that are involved in the remodelling process in tissue repair are presented.

Fibroblasts

Cytologically, fibroblasts are defined as elongated cells (Paper 4) with a typical marked rough endoplasmic reticulum and a typical Golgi apparatus that are common in cells that produce ECM and collagen (63) (Figure 4). The number of ECM molecules produced by fibroblasts are almost countless. The connective tissue from the fibroblasts surrounds the cells and provides a lattice to which the cells can bind and move along. Fibroblasts are in many cases a heterogeneous cell population and their behaviour is strongly dependent on and modulated by the surrounding environment, growth factors, and chemokines bound to the ECM.

10m

Figure 4. One of the structural cell types involved in tissue repair is the fibroblast. Fibroblasts are defined as elongated, cells with a typically marked rough endoplasmic reticulum. They can produce a number of extracellular matrix molecules. This cultured fibroblast was visualized by electron microscope.

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Fibroblasts are involved in tissue repair and wound healing because of their ability to produce ECM and collagen. The fibroblast can also use its contractile forces to diminish the area of the wound, and this property is enhanced if the cells are primed by -smooth muscle cells (-SMA). Sub-populations of fibroblasts seem to be more or less primed for specific functions; for example, Westergren-Thorsson

et al. have shown how fibroblast clones are negatively correlated to proliferation

and synthesis of decorin (64) and in fibroblasts from lung-transplanted patients there is a negative correlation between proteoglycan production and proliferation / migration (Paper 3).

Fibroblasts can be characterised using antibodies to fibroblast markers. Many of these markers overlap with those of macrophages and smooth muscle cells. However, instead combinations of markers can be used to identify these cells (Papers 3 and 4).

Myofibroblasts

A myofibroblast is a fibroblast phenotype that is activated, is more contractile, and has a higher production of ECM and collagen than fibroblasts. The shape of the cells is described as spindle-shaped. The phenotype was first observed by Gabbiani et al. in 1971 (65). Today, the myofibroblasts are well-characterised as -SMA-expressing cells. However, the pattern of expression is more complex than in smooth muscle cells, however, concerning both conserning enhancers and inhibitors in the downstream regulation of intracellular signalling (66;67).

There are two distinct populations of myofibroblasts, proto-myofibroblasts and differentiated myofibroblasts. Proto-myofibroblasts are formed after mechanical stress in fibroblasts. The characteristics of proto-myofibroblasts are that they express intracellular stress fibers and cell-surface fibronectin, which can both generate contractile forces. The differentiated myofibroblast develops from the proto-myofibroblast after TGF- stimulation, which increases the amount of fibronectin at the cell surface to form fibrils; after further mechanical stress, -SMA is formed intracellularly and focal adhesion at the cell surface (68).

Myofibroblasts are often located in areas of fibrosis, or in areas close to fibroblastic foci in IPF patients (69). They are capable of destroying overlying epithelial cells by secreting H2O2. The end result of the wound healing is

determined by whether or not the myofibroblast is capable of apoptosis. Without apoptosis, fibrosis develops and this process is driven by TGF- (70). One possible treatment for TGF--induced activation of myofibroblasts is interferon  (IFN-), which regulates TGF- by up-regulation of Smad7 through phosphorylation of signal transducers and activator of transcription (STAT) (71).

Fibroblasts are involved in tissue repair and wound healing because of their ability to produce ECM and collagen. The fibroblast can also use its contractile forces to diminish the area of the wound, and this property is enhanced if the cells are primed by -smooth muscle cells (-SMA). Sub-populations of fibroblasts seem to be more or less primed for specific functions; for example, Westergren-Thorsson

et al. have shown how fibroblast clones are negatively correlated to proliferation

and synthesis of decorin (64) and in fibroblasts from lung-transplanted patients there is a negative correlation between proteoglycan production and proliferation / migration (Paper 3).

Fibroblasts can be characterised using antibodies to fibroblast markers. Many of these markers overlap with those of macrophages and smooth muscle cells. However, instead combinations of markers can be used to identify these cells (Papers 3 and 4).

Myofibroblasts

A myofibroblast is a fibroblast phenotype that is activated, is more contractile, and has a higher production of ECM and collagen than fibroblasts. The shape of the cells is described as spindle-shaped. The phenotype was first observed by Gabbiani et al. in 1971 (65). Today, the myofibroblasts are well-characterised as -SMA-expressing cells. However, the pattern of expression is more complex than in smooth muscle cells, however, concerning both conserning enhancers and inhibitors in the downstream regulation of intracellular signalling (66;67).

There are two distinct populations of myofibroblasts, proto-myofibroblasts and differentiated myofibroblasts. Proto-myofibroblasts are formed after mechanical stress in fibroblasts. The characteristics of proto-myofibroblasts are that they express intracellular stress fibers and cell-surface fibronectin, which can both generate contractile forces. The differentiated myofibroblast develops from the proto-myofibroblast after TGF- stimulation, which increases the amount of fibronectin at the cell surface to form fibrils; after further mechanical stress, -SMA is formed intracellularly and focal adhesion at the cell surface (68).

Myofibroblasts are often located in areas of fibrosis, or in areas close to fibroblastic foci in IPF patients (69). They are capable of destroying overlying epithelial cells by secreting H2O2. The end result of the wound healing is

determined by whether or not the myofibroblast is capable of apoptosis. Without apoptosis, fibrosis develops and this process is driven by TGF- (70). One possible treatment for TGF--induced activation of myofibroblasts is interferon  (IFN-), which regulates TGF- by up-regulation of Smad7 through phosphorylation of signal transducers and activator of transcription (STAT) (71).

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The Origin of the Fibroblast/Myofibroblast

A few years ago, tissue-resident fibroblasts were thought to be the only possible origin of fibroblasts. In this thesis, both paper 1 and paper 2 focuses on fibrocytes; these are one of the more recently discovered origins of fibroblasts. Epithelial-mesenchymal transition and endothelial-Epithelial-mesenchymal transition are also known to be possible sources of fibroblasts. In bleomycin-induced lung fibrosis, one-third of the fibroblasts are derived from epithelium and one-fifth are derived from bone marrow. In this study no other origins were investigated, and conclusions concerning endothelial-mesenchymal transition and other origins are still unclear (72).

Resident Fibroblasts

As early as 1990, Darby et al. showed how resident fibroblasts proliferate and differentiate into myofibroblasts in an open scar wound healing model in the rat. The processes started 6 days after scar formation, with the highest level of fibroblasts 15 days later, and ended with apoptosis on days 20–25 after scar formation (73). Furthermore, fibroblasts from fibrotic patients have a higher proliferation capacity than those from healthy controls (64). Proliferation of the resident fibroblasts is probably driven both by factors in the tissue such as ECM molecules and by the inflammation.

Fibrocytes

Fibrocytes are progenitor cells that originate in the bone marrow. The fibrocytes have many characteristics that make them a discrete cell population. They are mostly known to have an important role in fibrotic lung diseases such as asthma (74;75), COPD, IPF (76) (Paper 1), and OB (Paper 2), but also in skin wound healing (77) and kidney fibrosis (78). Unfortunately, we do not have any specific marker for fibrocytes; instead, a combination of markers for different cell types is being used such as combining haematopoietic markers with mesenchymal markers. For example, there are molecules specific for leukocytes (CD45), monocytes (CD11a, CD11b, CD13), and stem cells (CD34), and also chemokine receptors (CXCR4), major histocompatibility complex (MHC) molecules, and mesenchymal markers (prolyl 4-hydroxylase, -SMA) (77;79). One of the most potent markers is CXCR4, which is expressed by 90% of the circulating pool of fibrocytes (80). The expression of these specific proteins changes as the fibrocytes are released from the bone marrow and recruited to the tissue. Mori et al. isolated circulating fibrocytes from mice and analysed the cells regarding their CD13, CD34, CD45, collagen I, and -SMA expression for one week in serum-free medium or in medium supplemented with TGF-, a factor involved in wound healing. The expression of CD13, CD34, and CD45 became reduced while the expression of collagen I was constantly high and the expression of -SMA increased. The differences were even higher when TGF- was present (81).

The Origin of the Fibroblast/Myofibroblast

A few years ago, tissue-resident fibroblasts were thought to be the only possible origin of fibroblasts. In this thesis, both paper 1 and paper 2 focuses on fibrocytes; these are one of the more recently discovered origins of fibroblasts. Epithelial-mesenchymal transition and endothelial-Epithelial-mesenchymal transition are also known to be possible sources of fibroblasts. In bleomycin-induced lung fibrosis, one-third of the fibroblasts are derived from epithelium and one-fifth are derived from bone marrow. In this study no other origins were investigated, and conclusions concerning endothelial-mesenchymal transition and other origins are still unclear (72).

Resident Fibroblasts

As early as 1990, Darby et al. showed how resident fibroblasts proliferate and differentiate into myofibroblasts in an open scar wound healing model in the rat. The processes started 6 days after scar formation, with the highest level of fibroblasts 15 days later, and ended with apoptosis on days 20–25 after scar formation (73). Furthermore, fibroblasts from fibrotic patients have a higher proliferation capacity than those from healthy controls (64). Proliferation of the resident fibroblasts is probably driven both by factors in the tissue such as ECM molecules and by the inflammation.

Fibrocytes

Fibrocytes are progenitor cells that originate in the bone marrow. The fibrocytes have many characteristics that make them a discrete cell population. They are mostly known to have an important role in fibrotic lung diseases such as asthma (74;75), COPD, IPF (76) (Paper 1), and OB (Paper 2), but also in skin wound healing (77) and kidney fibrosis (78). Unfortunately, we do not have any specific marker for fibrocytes; instead, a combination of markers for different cell types is being used such as combining haematopoietic markers with mesenchymal markers. For example, there are molecules specific for leukocytes (CD45), monocytes (CD11a, CD11b, CD13), and stem cells (CD34), and also chemokine receptors (CXCR4), major histocompatibility complex (MHC) molecules, and mesenchymal markers (prolyl 4-hydroxylase, -SMA) (77;79). One of the most potent markers is CXCR4, which is expressed by 90% of the circulating pool of fibrocytes (80). The expression of these specific proteins changes as the fibrocytes are released from the bone marrow and recruited to the tissue. Mori et al. isolated circulating fibrocytes from mice and analysed the cells regarding their CD13, CD34, CD45, collagen I, and -SMA expression for one week in serum-free medium or in medium supplemented with TGF-, a factor involved in wound healing. The expression of CD13, CD34, and CD45 became reduced while the expression of collagen I was constantly high and the expression of -SMA increased. The differences were even higher when TGF- was present (81).

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Fibrocytes have a role in many steps of angiogenesis. For example, fibrocytes express MMP-9, which helps these cells to penetrate the basement membrane during formation of new vessels. Isolated fibrocytes produce a number of pro-angiogenic factors such as bFGF, VEGF, granulocyte-macrophage stimulating factor (GM-CSF), interleukin-1 (IL-1), IL-8, and macrophage colony-stimulating factor (M-CSF). These factors induce migration, proliferation, and alignment of endothelial cells into tube-like structures (82).

Fibrocytes differ from fibroblasts in many ways. An immunologically important feature is antigen presentation. They express both MHC class I and class II antigens and co-factors CD80 and CD86. Furthermore, fibrocytes can migrate to lymphatic organs and sensitise naive T-cells. Previously, this feature was only thought to be a task of dendritic cells (83).

Recruitment of Fibrocytes

Fibrocytes have to be recruited from the bone marrow to the injured tissue, and one of the possibilities for recruitment is the CXCR4 – SDF-1 / CXCL12 axis. SDF-1 / CXCL12 belongsto the CXC family and has a length of 68 amino acids (8 kDa). The only receptor for SDF-1 / CXCL12 is the G-protein-coupled seven-span transmembrane receptor CXCR4 (84), which is present on its target cell. Binding causes changes to the cell: increased secretion of MMPs, VEGF, and NO, and also cytoskeletal rearrangements, which give increased mortality and chemotaxis. Some molecules involved in inflammation (hyaluronan, fibronectin, and fibrinogen) appear to increase the sensitivity to SDF-1 / CXCL12 (85).

The expression of CXCR4 and its ligand SDF-1 / CXCL12 is known to be up-regulated under hypoxic conditions by hypoxia-induced factor 1 (HIF-1) (86;87). The bone marrow is hypoxic compared to the surrounding vessels, and bone marrow has expression of SDF-1 / CXCL12. An injury in the lung leads to increased levels of SDF-1 / CXCL12 in the plasma (Paper 1), and fibrocytes are released from the bone marrow to migrate over a chemotactic gradient to the injured lung, where SDF-1 / CXCL12 is being expressed (88).

The importance of the CXCR4 – SDF-1 / CXCL12 axis has been shown by Phillips et al. using an animal model of lung fibrosis. In a bleomycin-induced model of lung fibrosis mice were treated with anti-CXCL12 antibodies and there was a significantly lower level of collagen and -SMA compared to the animals treated with control antibodies (89) (Figure 5). Another possible way of recruitment is a gradient of the chemokine secondary lymphoid tissue chemokine /

chemokine ligand 21 (SLC / CCL21), which is expressed in lymphoid organs and also in lung tissue under inflammatory conditions. The receptor is CCR7, but it is only expressed by less than 10% of circulating fibrocytes (80). This way of

Fibrocytes have a role in many steps of angiogenesis. For example, fibrocytes express MMP-9, which helps these cells to penetrate the basement membrane during formation of new vessels. Isolated fibrocytes produce a number of pro-angiogenic factors such as bFGF, VEGF, granulocyte-macrophage stimulating factor (GM-CSF), interleukin-1 (IL-1), IL-8, and macrophage colony-stimulating factor (M-CSF). These factors induce migration, proliferation, and alignment of endothelial cells into tube-like structures (82).

Fibrocytes differ from fibroblasts in many ways. An immunologically important feature is antigen presentation. They express both MHC class I and class II antigens and co-factors CD80 and CD86. Furthermore, fibrocytes can migrate to lymphatic organs and sensitise naive T-cells. Previously, this feature was only thought to be a task of dendritic cells (83).

Recruitment of Fibrocytes

Fibrocytes have to be recruited from the bone marrow to the injured tissue, and one of the possibilities for recruitment is the CXCR4 – SDF-1 / CXCL12 axis. SDF-1 / CXCL12 belongsto the CXC family and has a length of 68 amino acids (8 kDa). The only receptor for SDF-1 / CXCL12 is the G-protein-coupled seven-span transmembrane receptor CXCR4 (84), which is present on its target cell. Binding causes changes to the cell: increased secretion of MMPs, VEGF, and NO, and also cytoskeletal rearrangements, which give increased mortality and chemotaxis. Some molecules involved in inflammation (hyaluronan, fibronectin, and fibrinogen) appear to increase the sensitivity to SDF-1 / CXCL12 (85).

The expression of CXCR4 and its ligand SDF-1 / CXCL12 is known to be up-regulated under hypoxic conditions by hypoxia-induced factor 1 (HIF-1) (86;87). The bone marrow is hypoxic compared to the surrounding vessels, and bone marrow has expression of SDF-1 / CXCL12. An injury in the lung leads to increased levels of SDF-1 / CXCL12 in the plasma (Paper 1), and fibrocytes are released from the bone marrow to migrate over a chemotactic gradient to the injured lung, where SDF-1 / CXCL12 is being expressed (88).

The importance of the CXCR4 – SDF-1 / CXCL12 axis has been shown by Phillips et al. using an animal model of lung fibrosis. In a bleomycin-induced model of lung fibrosis mice were treated with anti-CXCL12 antibodies and there was a significantly lower level of collagen and -SMA compared to the animals treated with control antibodies (89) (Figure 5). Another possible way of recruitment is a gradient of the chemokine secondary lymphoid tissue chemokine /

chemokine ligand 21 (SLC / CCL21), which is expressed in lymphoid organs and also in lung tissue under inflammatory conditions. The receptor is CCR7, but it is only expressed by less than 10% of circulating fibrocytes (80). This way of

(27)

recruitment has been mentioned most in papers on renal fibrosis (77;78). The third way of recruitment of fibrocytes, the CCR2 / CCL2 axis, is only present in animals and has been shown to occur in lung tissue after injury (90).

SDF-1 / CXCL12

Fibrocytes

CXCR4

Figure 5. One possible way of recruitment of fibrocytes is by the CXCR4 – SDF-1 / CXCL12 axis. Fibrocytes that express CXCR4 originate in the bone marrow. A lung injury results in increased levels of SDF-1 / CXCL12, thus causing build-up of a gradient to recruit CXCR4-positive fibrocytes to the location of injury of the tissue.

Fibrocyte Differentiation

When fibrocytes have entered an injured tissue, they move through a matrix where many cytokines are bound. These cytokines are known to influence the behaviour of the fibrocytes; as mentioned previously, SDF-1 induces migration by interacting with CXCR4. Mori et al. showed how TGF- is capable of inducing fibrocytes to differentiate into -SMA-positive myofibroblasts (81). The pathways that are involved in this differentiation are activation of Smad 2/3 and stress-activated protein kinase / jun N-terminal kinases mitogen-activated protein kinases (SAPK/JNK MAPK) (91). The markers on the fibrocytes change during recruitment to the injured tissue. The expression of mesenchymal markers increases while the expression of haematopoietic markers decreases (92).

Another possible way of differentiation of fibrocytes is to adipocytes, which is driven by specific adipogenetic hormones and cytokines and which follows activation of specific adipocyte genes. On the other hand, TGF- inhibits this

recruitment has been mentioned most in papers on renal fibrosis (77;78). The third way of recruitment of fibrocytes, the CCR2 / CCL2 axis, is only present in animals and has been shown to occur in lung tissue after injury (90).

SDF-1 / CXCL12

Fibrocytes

CXCR4

Figure 5. One possible way of recruitment of fibrocytes is by the CXCR4 – SDF-1 / CXCL12 axis. Fibrocytes that express CXCR4 originate in the bone marrow. A lung injury results in increased levels of SDF-1 / CXCL12, thus causing build-up of a gradient to recruit CXCR4-positive fibrocytes to the location of injury of the tissue.

Fibrocyte Differentiation

When fibrocytes have entered an injured tissue, they move through a matrix where many cytokines are bound. These cytokines are known to influence the behaviour of the fibrocytes; as mentioned previously, SDF-1 induces migration by interacting with CXCR4. Mori et al. showed how TGF- is capable of inducing fibrocytes to differentiate into -SMA-positive myofibroblasts (81). The pathways that are involved in this differentiation are activation of Smad 2/3 and stress-activated protein kinase / jun N-terminal kinases mitogen-activated protein kinases (SAPK/JNK MAPK) (91). The markers on the fibrocytes change during recruitment to the injured tissue. The expression of mesenchymal markers increases while the expression of haematopoietic markers decreases (92).

Another possible way of differentiation of fibrocytes is to adipocytes, which is driven by specific adipogenetic hormones and cytokines and which follows activation of specific adipocyte genes. On the other hand, TGF- inhibits this

References

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